KR102166453B1 - Composition for preventing or treating pulmonary diseases comprising hapln1 - Google Patents

Abstract

The present invention relates to novel recombinant human hyaluronan and proteoglycan link protein 1 (HAPLN1), and a composition for preventing or treating lung diseases which comprises the novel recombinant human hyaluronan as an active ingredient. The recombinant human HAPLN1 according to the present invention has an excellent effect of alleviating damage to alveolar cells due to aging or a decrease in elastin. Thus, lung diseases such as chronic bronchitis, asthma, pulmonary emphysema, and chronic obstructive pulmonary diseases can be effectively prevented or treated.

Description

Composition for preventing or treating lung diseases, including HAPLN1 {COMPOSITION FOR PREVENTING OR TREATING PULMONARY DISEASES COMPRISING HAPLN1}

The present invention relates to a novel recombinant human HAPLN1 (hyaluronan and proteoglycan link protein 1) protein, and a composition for preventing or treating lung diseases comprising the same as an active ingredient.

Chronic obstructive pulmonary diseases (COPD), the fourth leading cause of death in the world, suffered from COPD as of 2016, with approximately 251 million people (4.8% of the total population). It is reported that about 3.17 million people died from COPD. It is also more common in men than in women, and the number of patients with chronic obstructive pulmonary disease is increasing every year.

Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease that occurs more frequently than lung cancer. It is characterized by continuous progression of respiratory symptoms due to cough, sputum, and dyspnea and irreversible airway obstruction. It is widely known that the main causes of COPD are smoking, occupational exposure, and air pollution such as fine dust. Due to these various causes, small airway lung disease and pulmonary parenchymal destruction act in combination, causing airflow limitation, leading to various clinical symptoms.

According to recent studies, aging itself can be an important cause of COPD and has been shown to be a risk factor. In particular, it rapidly increases from the 50s to the later age, and the incidence rate in the elderly aged 60 or older is 2-3 times higher than that of the younger generation. COPD has a profound effect on the lungs, and in the process, damage to the alveoli occurs, and air exchange is not smoothly performed due to the damage to the alveoli. It is known that the reason why the disease does not occur while smoking in the younger generation is due to active recovery and regeneration as well as damage.

Along with the global aging trend, cell senescence, shorter telomeres, and a decrease in some anti-aging molecules cause chronic inflammatory reactions in the lungs, resulting in narrowing of the small airways and elastin in the lung tissue. It can be a direct cause of emphysema, which is known to appear as a result of the destruction of the back. The pathogenesis and biochemical changes of these senile emphysema are very similar to those of common emphysema. Since emphysema is not reversible but irreversible like asthma or bronchitis, it is very difficult to treat and there are no therapeutic or preventive drugs that can be effectively treated.

In particular, elastin protein, a constituent of the extracellular matrix (ECM), is a very important protein that provides elasticity and elasticity that are essential for the gas exchange function of the alveoli. As a result, when the alveolar wall is destroyed or damaged, the alveolar wall becomes unrecoverable, causing the alveolar dilatation-constriction action, that is, breathing problems, and eventually proceeds to COPD.

As COPD is becoming an important global issue with the aging trend, the question of whether it is actually possible to treat and prevent COPD caused by aging is questioned, as the lungs were known as an organ that cannot recover or regenerate. The possibility has been perceived as very slim. However, since 2012 proved that adult lung tissue is an organ that can be newly regenerated by a study by Butler et al., several very recent studies have shown that a group of lung epithelial cells have the ability to self-differentiate and proliferate, resulting in damaged or degenerated alveoli. It is expected that therapeutic and preventive medicines will be more actively studied in the future along with the advancement of technology for regeneration and recovery of degenerated alveoli by revealing that it can recover or regenerate regeneration through cellular biological evidence and mechanisms.

As described above, COPD is a lung disease characterized by irreversible airflow limitation, which occurs due to damage to the airways and lung parenchyma due to chronic inflammation and continues to progress, but is known as a disease that can be prevented and treated as described above. Currently, the most frequently used COPD treatments worldwide are inhaled corticosteroids (ICS), long-acting muscarinic antagonists (LAMA), and long-acting beta agonists (long-acting) asthma treatments. There are treatments for chronic bronchitis and emphysema, such as acting β-agonist (LABA), but these treatments do not completely cure COPD. In particular, inhaled steroids exert a strong partial anti-inflammatory effect because drugs are administered directly into the airways, but systemic side effects may increase when steroids administered into the airways are transported from the alveoli to the blood through the airways. have. The side effects of these existing products may cause patients to feel anxious, and in severe cases, it may lead to refusal to administer administration, so efforts to develop drugs with less side effects are needed.

Republic of Korea Patent Publication No. 10-2007-0112086 (published on November 22, 2007)

In order to solve the above problems, the present invention provides a novel recombinant protein and an excellent composition for preventing or treating lung diseases containing the same as an active ingredient.

The novel recombinant protein according to the present invention may be a recombinant human HAPLN1 (hyaluronan and proteoglycan link protein 1) protein having the amino acid sequence of SEQ ID NO: 1.

The pharmaceutical composition for preventing or treating lung diseases according to the present invention may contain the recombinant human HAPLN1 protein as an active ingredient.

The health functional food composition for preventing or improving lung diseases according to the present invention may contain the recombinant human HAPLN1 protein as an active ingredient.

The recombinant human HAPLN1 protein according to the present invention has an excellent effect of improving alveolar damage due to aging or elastin reduction, and prevents lung diseases such as chronic bronchitis, asthma, emphysema, and chronic obstructive pulmonary disease caused by aging or elastin reduction. Or it can be used as a therapeutic pharmaceutical composition, a health functional food composition, and the like.

The composition can safely and effectively prevent or treat the lung disease, which is increasing every year with the global aging trend, since there are few side effects caused by the existing drugs for treating lung diseases.

1 is an alveolar test result of an elderly mouse according to an experimental example of the present invention, FIG. 1A is an image observed under a microscope after staining the alveolar tissue, and FIGS. 1B and 1C are graphs quantifying the density of the alveoli. Here,'Young-saline' is a group administered phosphate buffered saline (PBS) to a normal control group (Young group), and'Old-saline' is a group administered PBS to an elderly control group (Old group), 'Old-rhHAPLN1' refers to a group administered with PBS to an elderly rhHAPLN1 administration group (Old + rhHAPLN1 group).
2 is a schematic diagram of a spray injection (aerosolization) method according to an experimental example of the present invention.
3 is an experimental result of an aerosol inhalation (AH group) according to an experimental example of the present invention, 3A is an image obtained by staining alveolar tissue for each group and observed under a microscope, and 3B and 3C are MLI (mean This is a graph of the result of measuring linear intercepts) values.
4 is an experimental result of an intratracheal instillation (TI group) according to an experimental example of the present invention, 4A is an image obtained by staining alveolar tissue for each group and observed with a microscope, and 4B and 4C are MLI thereof. This is a graph of the result of measuring the (mean linear intercepts) value.

Hereinafter, the present invention will be described in detail.

The present inventors have completed the present invention by producing a novel recombinant HAPLN1 protein, and confirming the effect of improving alveolar damage and its therapeutic effect in an animal model of chronic obstructive pulmonary disease.

In the present specification, "prevention" means any action of inhibiting the occurrence of or delaying the onset of lung disease, or at least one symptom of the disease by administration of the pharmaceutical composition or health functional food composition according to the present invention. . In addition, it includes treatment of a subject with remission of the disease to prevent or prevent recurrence.

In the present specification, the term "treatment" refers to any act of improving or beneficially altering the symptoms, such as alleviating, reducing, or eliminating at least one symptom of a lung disease or the disease by administration of the pharmaceutical composition according to the present invention. Means.

In the present specification, "improvement" refers to a lung disease, or at least one symptom of the disease by ingestion of the health functional food composition according to the present invention, alleviating, reducing, or eliminating the symptoms, such as improving or advantageously It means all actions.

In the present specification, the "pharmaceutical composition" means a composition administered for a specific purpose, and for the purposes of the present invention, it is administered to prevent or treat a lung disease or at least one symptom of the disease.

In the present specification, the term "health functional food" includes foods manufactured and processed using raw materials or ingredients having functions useful for the human body according to the Health Functional Food Act No. 6727, and in addition to supplying nutrition, the object of the present invention It refers to medicines and foods with high medical effects that have been processed to efficiently exhibit biomodulatory functions such as prevention or improvement of upper lung disease, body defense, immunity, and recovery.

The present invention provides a recombinant human HAPLN1 (hyaluronan and proteoglycan link protein 1) protein having the amino acid sequence of SEQ ID NO: 1.

The "HAPLN1 protein" is a protein that stabilizes hyaluronic acid by linking hyaluronic acid to proteoglycans, and is a constituent protein in the extracellular matrix first discovered in joints of vertebrates.

The "recombinant human HAPLN1 protein" according to the present invention is a novel recombinant protein prepared using the HAPLN1 protein.

The present invention provides a pharmaceutical composition for preventing or treating lung diseases containing a recombinant human HAPLN1 protein having an amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The recombinant human HAPLN1 protein may have an effect of improving alveolar damage due to aging or elastin reduction, and thus may be used as a pharmaceutical composition for preventing or treating lung diseases. In addition, according to an experimental example of the present invention, it can be confirmed that a significant therapeutic effect is shown in an animal model of chronic obstructive pulmonary disease.

In the pharmaceutical composition according to the present invention, the lung disease is one or more selected from the group consisting of chronic bronchitis, asthma, emphysema, and chronic obstructive pulmonary diseases (COPD). However, the present invention is not limited thereto, and may include various diseases caused by lung damage.

The pharmaceutical composition according to the present invention can be prepared according to a conventional method in the pharmaceutical field. The pharmaceutical composition may be blended with an appropriate pharmaceutically acceptable carrier depending on the formulation in addition to the active ingredient, and further includes excipients, diluents, dispersants, emulsifiers, buffers, stabilizers, binders, disintegrants, solvents, etc., as needed. It can be manufactured. The appropriate carrier, etc., does not inhibit the activity and properties of the recombinant human HAPLN1 protein according to the present invention, and may be selected differently depending on the dosage form and formulation.

The pharmaceutical composition according to the present invention may be applied in any dosage form, and more particularly, may be formulated and used in parenteral dosage forms of oral dosage forms, external preparations, suppositories and sterile injectable solutions according to conventional methods.

Among the oral dosage forms, the solid dosage form may be in the form of tablets, pills, powders, granules, capsules, etc., and at least one excipient such as starch, calcium carbonate, sucrose, lactose, sorbitol, mannitol, cellulose, gelatin, etc. It can be prepared by mixing, and in addition to simple excipients, lubricants such as magnesium stearate and talc may also be included. In addition, in the case of the capsul formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be further included.

Among the oral dosage forms, liquid dosage forms correspond to suspensions, liquid solutions, emulsions, syrups, and the like.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. may be included. have.

The parenteral formulation may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used as the non-aqueous solvent and suspension. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used. The present invention is not limited thereto, and any suitable agent known in the art may be used.

In addition, the pharmaceutical composition according to the present invention may further include calcium or vitamin D ₃ to improve therapeutic efficacy.

The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.

In the present specification, "a pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects.

The effective dosage level of the pharmaceutical composition is the purpose of use, the patient's age, sex, weight and health condition, the type of disease, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the administration time, the administration route and the rate of excretion, the treatment It may be determined differently depending on the duration, factors including drugs used in combination or concurrently and other factors well known in the medical field. For example, although not constant, generally 0.001 to 100 mg/kg, preferably 0.01 to 10 mg/kg may be administered once to several times a day. The above dosage does not in any way limit the scope of the present invention.

The pharmaceutical composition according to the present invention may be administered to any animal that may cause lung disease, and the animal may include, for example, humans and primates as well as livestock such as cattle, pigs, horses, and dogs. .

The pharmaceutical composition according to the present invention can be administered by an appropriate route of administration according to the form of the formulation, and can be administered through various routes, either oral or parenteral, as long as it can reach the target tissue. The method of administration is not particularly limited, for example, oral, rectal or intravenous, intramuscular, skin application, subcutaneous, respiratory inhalation, intrauterine dura mater or intracere-broventricular injection. Can be.

The pharmaceutical composition according to the present invention may be used alone to prevent or treat lung diseases, or may be used in combination with surgery or other drug treatment.

In addition, the present invention provides a health functional food composition for preventing or improving lung diseases containing a recombinant human HAPLN1 protein having the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The recombinant human HAPLN1 protein may have an effect of improving alveolar damage due to aging or elastin reduction, and thus may be used as a health functional food composition for preventing or improving lung diseases.

In the health functional food composition according to the present invention, the lung disease is selected from the group consisting of chronic bronchitis, asthma, emphysema, and chronic obstructive pulmonary diseases (COPD). It may be one or more, but is not limited thereto, and may include various diseases due to lung damage.

In the health functional food composition according to the present invention, the health functional food may be prepared as a powder, granule, tablet, capsule, syrup or beverage for the purpose of preventing or improving lung disease, and the form that the food can take There is no limitation, and it may include all foods in the usual sense. For example, beverages and various drinks, fruits and processed foods thereof (canned fruit, jam, etc.), fish, meat and processed foods thereof (ham, bacon, etc.), bread and noodles, cookies and snacks, dairy products (butter, cheese, etc.) ), and the like, and may include all functional foods in the usual sense. In addition, foods used as feed for animals may also be included.

The health functional food composition according to the present invention may be prepared by further comprising a food pharmaceutically acceptable food additive (food additive) and other appropriate auxiliary ingredients commonly used in the art.

Unless otherwise specified, the suitability as a food additive may be determined according to the standards and standards for the relevant item in accordance with the general rules and general test methods for food additives approved by the Food and Drug Administration. Examples of items listed in the'Food Additives Code' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned.

The other auxiliary ingredients are, for example, flavoring agents, natural carbohydrates, sweetening agents, vitamins, electrolytes, colorants, pectic acids, alginic acids, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents, etc. It may further contain. In particular, as the natural carbohydrates, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol may be used. , As the sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.

The effective dose of the recombinant HAPLN1 protein contained in the health functional food according to the present invention may be appropriately adjusted according to the purpose of use, such as preventing or improving lung diseases. The composition has the advantage of not having side effects that may occur during long-term use of general drugs by using food as a raw material, and is excellent in portability, and can be taken as an adjuvant for preventing or improving lung diseases.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Example 1> Production of recombinant human HAPLN1 protein (rhHAPLN1)

1. Amino acid sequence of recombinant human HAPLN1 protein

The amino acid sequence constituting the recombinant HAPLN1 protein is as follows.

DHLSDNYTLDHDRAIHIQAENGPHLLVEAEQAKVFSHRGGNVTLPCKFYRDPTAFGSGIHKIRIKWTKLTSDYLKEVDVFVSMGYHKKTYGGYQGRVFLKGGSDSDASLVITDLTLEDYGRYKCEVIEGLEDDTVVVALDLQGVVFPYFPRLGRYNLNFHEAQQACLDQDAVIASFDQLYDAWRGGLDWCNAGWLSDGSVQYPITKPREPCGGQNTVPGVRNYGFWDKDKSRYDVFCFTSNFNGRFYYLIHPTKLTYDEAVQACLNDGAQIAKVGQIFAAWKILGYDRCDAGWLADGSVRYPISRPRRRCSPTEAAVRFVGFPDKKHKLYGVYCFRAYN (SEQ ID NO: 1)

2. Expression, purification and storage of recombinant human HAPLN1 protein (rhHAPLN1)

For the production of recombinant human HAPLN1 (rhHAPLN1; gene number 2678736), a DNA vector encoding a recombinant human HAPLN1 amino acid sequence was infected with a host cell, Expi™ 293 cells (ThermoFisher Scientific Co., Waltham, MA, USA). transfection). For purification, secretion signal peptides, 10 histidines (H, His, histidine), and TEV protease-recognition sites were inserted into the amino terminal. After 3 days of vector infection, the culture medium was collected and purified through HisTrap column (GE Healthcare, IL, USA), and then the TEV protease-recognized sequence was cut with TEV protease, and molecules containing histidine were transferred to DynaBeads (Thermo Fisher Scientific). Removed using. The thus obtained solution was dialyzed against 40 mM Tris-HCl, 1 M NaCl, and pH 8.0 for 16 hours. The protein concentration of the final tablet is 0.11mg/ml, the solvent is 20mM Tris-HCl, 0.5M NaCl, pH 8.0, 50% glycerol, subdivided as a single dose, and stored in a refrigerator at -20℃ for use. I did.

<Experimental Example 1> Confirmation of the effect of improving alveolar structure in elderly mice by repeated intraperitoneal administration of recombinant human HAPLN1 protein (rhHAPLN1)

1. Preparation and rearing of experimental animals

As for the experimental animals, a 2-month-old male C57BL/6J (Young Bio Co., Ltd., Korea) mouse was set as a young mouse, and a 20-month-old male C57BL/6J mouse was set as an old mouse. Water and feed were allowed to be eaten freely, and the temperature in the breeding room was maintained at 21~24℃ and humidity at 40%~60%, and the day and night cycles were each 12 hours.

Five mice were assigned to the normal control group (Young group), and the elderly control group (Old group) and the elderly rhHAPLN1 group (Old + rhHAPLN1 group) were assigned 5 mice each. In the elderly rhHAPLN1 group (Old + rhHAPLN1 group), rhHAPLN1 diluted in phosphate buffered saline (PBS) was injected intraperitoneally (intraperitoneal injection; IP injection) as a dose of 0.1 mg/kg at a dose of 80 μl once. , This was done 3 times a week for a total of 3 weeks. The other two control groups were administered the same amount of PBS in the same manner.

2. Alveolar tissue staining and microscopic observation

After cardiac perfusion was performed at the end of the experiment, the left lung was bisected into upper and lower parts, fixed with neutral buffered 10% formalin (NBF), and paraffin tissue sections were prepared so that the cut surface of the upper piece was visible. Tissue sections were stained with hematoxylin & eosin (H&E) method, and photographed using a Ni-U (Nikon) microscope and a DS-Ri1 (Nikon) digital camera, and the results are shown in Fig. 1(A). ) (Scale bar = 100 μm).

In addition, for the stained tissue sections of each individual, mean linear intercepts (MLI) values were calculated using the Image J program to quantify the alveolar density. The higher the MLI value, the lower the alveolar surface area, and is used as an indicator of the degree of emphysema or chronic obstructive pulmonary diseases (COPD). The formula is Lm(μm) = number of intersections / (grid length(μm) × number of lines), and after measuring the average of 3 random spots per animal, Sigma plot 12.0 and Graph Pad Prism 8 were calculated for 5 animals. Statistical processing was performed using. The results are shown in Fig. 1(B).

1(A) and 1(B), the alveolar MLI value (μm) of aging mice increased by about 141% compared to that of young mice (p <0.001). Meanwhile, due to the cause of aging itself, this increase was found to be reduced by about 56% by treatment with rhHAPLN1 (p <0.0435).

<Experimental Example 2> Efficacy of recombinant human HAPLN1 protein (rhHAPLN1) in a chronic obstructive pulmonary disease (COPD) model induced by porcine pancreatic elastase (PPE)

1. Preparation and rearing of experimental animals

Experimental animals were 6 to 10 weeks old female C57BL6/N (Young Bio Co., Ltd., Republic of Korea) mice weighing 20-25 g, and the mice were divided into 5 mice for each group. Water and feed were allowed to be eaten freely, and the temperature in the breeding room was maintained at 21~24℃ and humidity at 40%~60%, and the day and night cycles were each 12 hours.

2. Induction of chronic obstructive pulmonary disease (COPD)

Suki et al (2017, Methods Mol. Biol. 1639:67-75) and Wright et al (2008, Am J Physiol Lung Mol Physiol 295: L1-L15) were referenced to prepare a COPD-induced mouse model.

In order to inhale elastase into the lungs, first, porcine pancreatic elastase (EC134; porcine pancreatic elastase; PPE) was purchased from Elastin Products Company Inc. (Owensville, Missouri, USA) and 6IU/30μl per animal. Was administered once a day before drug treatment. The administration was divided into 2 groups according to the inhalation method.

Specifically, the aerosol inhalation group (AH) was administered through the oral cavity using a 20G (0.9×50mm) oral zonde needle. It was administered by carefully opening the mouth, pulling the tongue, dropping the elastase solution on the back, that is, on the distal oropharynx, and at the same time blocking both nostrils to induce inhalation. On the other hand, intratracheal instillation (TI) was administered by inducing spontaneous inhalation of mice by instilling in the nasal cavity using an Eppendorf pipette. The weight of the mice was measured twice a week, and each group was divided into 4 subgroups for administration of the composition drug.

3. Efficacy of pharmaceutical composition

Aerosol inhalation group (AH group) was again AH-1: No PPE treat (normal) group, AH-2: PPE + saline group, AH-3: PPE + rhHAPLN1 group, AH-4: PPE + hyaluronic acid (HA) Groups were divided, and the endotracheal inhalation group (TI group) was again TI-1: No PPE treat (normal) group, TI-2: PPE + saline group, TI-3: PPE + rhHAPLN1 group, TI-4: PPE + Re-divided into HA group. Here, HA is hyaluronic acid, and it is known to have an effect on COPD, so it was used as a positive control. For the basis of administration and determination of the concentration of HA, Cantor et al (2005, Experimental Lung Research, 31:417-430) were referred to, and accordingly, the Hyaluronic acid sodium salt derived from Streptococcus equi. ) (Sigma-Aldrich; St. Louis, MO, USA, Cat. 73641-10MG) was purchased and used.

Each pharmaceutical composition sample was used with a Mass Dosing System, and specifically, it was administered 5 times a week by aerosolization method from 1 day after elastase administration using an aerosol chamber (Data Science International) (Ball flow meter 3, aerosol). 1 hour, duty 30%). A schematic diagram illustrating the spray injection method is presented in FIG. 2.

Each sample was sprayed with a spray injection device at a concentration of 0.33% (w/v) rhHAPLN1, 5.71% (w/v) HA by adjusting to 6.8 ml per time using physiological saline as a solvent, and the first day of the last spray administration on the 21st day. Then, after performing heart perfusion under anesthesia, the left lung is bisected horizontally, the lower part is taken, fixed with neutral buffered 10% formalin (NBF), and a paraffin tissue section so that the cut surface of the upper piece is visible. Was produced. Tissue sections were stained with the hematoxylin & eosin (H&E) method, and photographed at 200 magnification using a Ni-U (Nikon) microscope and a DS-Ri1 (Nikon) digital camera, and alveolar tissue photo data ( Scale bar = 100 μm) was obtained and mean linear intercepts (MLI) of alveoli were measured. The higher the MLI value, the lower the alveolar surface area is and is used as an indicator of the degree of emphysema or COPD. The formula is Lm(μm) = number of intersections / (grid length(μm) × number of lines).After measuring the average value of 3 random animals per animal, Sigma plot 12.0 and Graph Pad Prism 8 were calculated for 5 animals. Statistical processing was performed using.

The experimental results of the aerosol inhalation (AH group) are shown in Fig. 3, and the experimental results of the intratracheal instillation (TI group) are shown in Fig. 4, respectively.

Figure 3 is a result of the aerosol inhalation (AH) group, referring to Figures 3A to 3C, the PPE-treated group (AH-2) 21.2 μm compared to the untreated normal group (AH-1) 13.8 μm It increased about 1.5 times (p <0.001). Compared to the PPE treatment group 21.2 μm, the rhHAPLN1 treatment group (AH-3) decreased by about 95% to 14.5 μm (p <0.00002), indicating that it recovered to the state of the almost normal group.

In contrast, the 5.71% (w/v) HA treatment group (AH-4), which was about 17 times higher than 0.33% (w/v) in terms of concentration, decreased to about 73% (p <0.002), and thus treated with the positive control HA. In terms of effect, it can be seen that the effect is slightly lower than about 95% of rhHAPLN1 treatment.

4 is an experimental result of an intratracheal instillation (TI group), and referring to FIGS. 4A to 4C, a PPE-treated group (TI-2) 22.1 μm was a normal group without treatment (TI-1). It increased about 1.6 times compared to 13.8 μm (p <0.001). Compared to the PPE treatment group (TI-2) 22.1 μm, the rhHAPLN1 treatment group (TI-3) was 16.1 μm, which was about 72% decrease (p <0.0009).

In contrast, the 5.71% (w/v) HA treatment group (TI-4), which was about 17 times higher than 0.33% (w/v) in terms of concentration, decreased to about 64% (p <0.00123), so that the positive control HA treatment It can also be seen that in terms of effect, it shows a slightly lower efficacy than about 72% of rhHAPLN1 treatment.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> COMPOSITION FOR PREVENTING OR TREATING PULMONARY DISEASES COMPRISING HAPLN1 <130> ADP-2019-0581 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 339 <212> PRT <213> Artificial Sequence <220> <223> Recombinant human HAPLN1 <400> 1 Asp His Leu Ser Asp Asn Tyr Thr Leu Asp His Asp Arg Ala Ile His 1 5 10 15 Ile Gln Ala Glu Asn Gly Pro His Leu Leu Val Glu Ala Glu Gln Ala 20 25 30 Lys Val Phe Ser His Arg Gly Gly Asn Val Thr Leu Pro Cys Lys Phe 35 40 45 Tyr Arg Asp Pro Thr Ala Phe Gly Ser Gly Ile His Lys Ile Arg Ile 50 55 60 Lys Trp Thr Lys Leu Thr Ser Asp Tyr Leu Lys Glu Val Asp Val Phe 65 70 75 80 Val Ser Met Gly Tyr His Lys Lys Thr Tyr Gly Gly Tyr Gln Gly Arg 85 90 95 Val Phe Leu Lys Gly Gly Ser Asp Ser Asp Ala Ser Leu Val Ile Thr 100 105 110 Asp Leu Thr Leu Glu Asp Tyr Gly Arg Tyr Lys Cys Glu Val Ile Glu 115 120 125 Gly Leu Glu Asp Asp Thr Val Val Val Ala Leu Asp Leu Gln Gly Val 130 135 140 Val Phe Pro Tyr Phe Pro Arg Leu Gly Arg Tyr Asn Leu Asn Phe His 145 150 155 160 Glu Ala Gln Gln Ala Cys Leu Asp Gln Asp Ala Val Ile Ala Ser Phe 165 170 175 Asp Gln Leu Tyr Asp Ala Trp Arg Gly Gly Leu Asp Trp Cys Asn Ala 180 185 190 Gly Trp Leu Ser Asp Gly Ser Val Gln Tyr Pro Ile Thr Lys Pro Arg 195 200 205 Glu Pro Cys Gly Gly Gln Asn Thr Val Pro Gly Val Arg Asn Tyr Gly 210 215 220 Phe Trp Asp Lys Asp Lys Ser Arg Tyr Asp Val Phe Cys Phe Thr Ser 225 230 235 240 Asn Phe Asn Gly Arg Phe Tyr Tyr Leu Ile His Pro Thr Lys Leu Thr 245 250 255 Tyr Asp Glu Ala Val Gln Ala Cys Leu Asn Asp Gly Ala Gln Ile Ala 260 265 270 Lys Val Gly Gln Ile Phe Ala Ala Trp Lys Ile Leu Gly Tyr Asp Arg 275 280 285 Cys Asp Ala Gly Trp Leu Ala Asp Gly Ser Val Arg Tyr Pro Ile Ser 290 295 300 Arg Pro Arg Arg Arg Cys Ser Pro Thr Glu Ala Ala Val Arg Phe Val 305 310 315 320 Gly Phe Pro Asp Lys Lys His Lys Leu Tyr Gly Val Tyr Cys Phe Arg 325 330 335 Ala Tyr Asn

Claims (6)

delete As a pharmaceutical composition for preventing or treating lung diseases containing a recombinant human HAPLN1 (hyaluronan and proteoglycan link protein 1) protein consisting of an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient,
The lung disease is chronic bronchitis (chronic bronchitis), asthma (asthma), emphysema (emphysema) and chronic obstructive pulmonary disease (COPD), characterized in that at least one selected from the group consisting of a pharmaceutical composition for preventing or treating pulmonary disease. The method of claim 2,
The recombinant human HAPLN1 protein,
A pharmaceutical composition for preventing or treating lung diseases, characterized in that to improve alveolar damage caused by aging or elastin reduction. delete As a health functional food composition for preventing or improving lung diseases containing a recombinant human HAPLN1 protein consisting of an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient,
The lung disease is one or more selected from the group consisting of chronic bronchitis, asthma, emphysema, and chronic obstructive pulmonary disease (COPD). . delete

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